Chronic inflammation decreases arcuate kisspeptin expression in male sheep.

Lipopolysaccharide (LPS) from Gram-negative bacteria induces an immune response and impairs reproduction through suppression of gonadotropin releasing hormone (GnRH), subsequently luteinizing hormone (LH) secretion. While there is evidence that acute inflammation inhibits kisspeptin, little is known about the impact of chronic inflammation on this key reproductive neuropeptide in livestock species. Thus, we sought to examine a central mechanism whereby LPS suppresses LH secretion in sheep. Twenty wethers were randomly assigned to one of five treatment groups: control (CON; n=4), single acute IV LPS dose (SAD; n=4), daily acute IV LPS dose (DAD; n=4), daily increasing IV LPS dose (DID; n=4), and chronic subcutaneous LPS dose (CSD; n=4). On Days 1 and 7, blood samples were collected every 12 minutes for 360 minutes using jugular venipuncture. Following blood collection on Day 7, all animals were euthanized, brain tissue was perfused with 4% paraformaldehyde, and hypothalamic blocks were removed and processed for immunohistochemistry. On Day 1, LH pulse frequency was significantly lower (p=0.02) in SAD (0.25 ± 0.1 pulses/hour), DAD (0.25 ± 0.1 pulses/hour), DID (0.35 ± 0.1 pulses/hour), and CSD (0.40 ± 0.1 pulses/hour) compared to CON (0.70 ±0.1 pulses/hour). On Day 7, only DID animals (0.35 ± 0.1 pulses/hour) had significantly lower (p=0.049) LH pulse frequency compared to controls (0.85 ± 0.1 pulse/hour). Furthermore, only DID animals (33.3 ± 10.9 cells/section/animal) had significantly fewer (p=0.001) kisspeptin-immunopositive cells compared to controls (82.6 ± 13.6 cells/section/animal). Taken together, we suggest that daily increasing doses of LPS is a powerful inhibitor of kisspeptin neurons in young male sheep and a physiologically relevant model to examine the impact of chronic inflammation on the reproductive axis in livestock.

Serum amyloid A, Serum Amyloid A1 and Haptoglobin in pregnant mares and their fetuses after experimental induction of placentitis.

Serum amyloid A (SAA) and Haptoglobin (Hp) are acute phase proteins, produced during inflammation, such as placentitis. In horses, SAA and SAA1 are protein coding genes. Objectives were to analyze SAA and Hp concentrations and relative abundance of SAA, SAA1 and Hp mRNA transcript in maternal and fetal tissues after experimental induction of placentitis or mares of a control group. Serum Amyloid A family proteins were in marked abundance in the stroma of the endometrium and chorioallantois associated with inflammatory cells. Maternal plasma SAA concentrations were greater (P = 0.01) in mares with experimentally induced placentitis compared to those of the control group. Maternal Hp from the groups were not different, but fetal Hp concentrations of mares with experimentally induced placentitis were greater (P = 0.02). Maternal plasma SAA and Hp concentrations were greater than fetal plasma concentrations in mares with experimentally induced placentitis (P < 0.05). Relative abundance of SAA mRNA transcript was greater in the maternal, fetal liver and chorioallantois of mares with experimentally induced placentitis (P < 0.05) compared to those in the control group. Interestingly, relative abundance of SAA1 mRNA transcript was greater in the chorioallantois of mares with experimentally induced placentitis (P < 0.05). The SAA and Hp concentrations, therefore, were greater in mares with induced placentitis. Furthermore, relative abundance of SAA1 mRNA transcript is specifically greater in the chorioallantois of mares with placentitis, which warrants further studies to elucidate the immunological response of SAA1 in the chorioallantois of mares with placentitis.

Changes in circulating concentrations of testosterone and estrone sulfate after human chorionic gonadotropin administration and subsequent to castration of 2-year-old stallions.

Reproductive steroids testosterone (T) and estrone sulfate (E1S) are used as diagnostic markers for cryptorchidism in horses. The human chorionic gonadotropin (hCG) stimulation test is used as a diagnostic aid because administration of this hormone results in greater incremental differences in circulating steroid concentrations. Thoughts regarding optimal sampling times following hCG administration, however, are inconsistent. Additionally, determination of half-life of these steroids is important in postsurgical samples to confirm complete removal of testicular tissue. Objectives of this study, therefore, were to determine optimal sampling periods for peak T and E1S after hCG administration and half-life of these steroids after castration. Eight pony stallions were randomly assigned to control or treatment groups (5000 IU hCG). Blood samples were collected following hCG administration. Subsequently, stallions were castrated and blood samples were collected post-castration. The T concentrations were greatest at 72 h after hCG and were greater (P < 0.02) in samples from hCG-treated than control animals: 9,903.4 ± 384 and 784.0 ± 192 pg/mL, respectively (Mean ± SEM). The T concentrations were also greater at 1, 12, 24, 48 and 96 h. The E1S concentrations did not change after administration of hCG. The T response to hCG administration was biphasic with a maximal response between 48-96 h after administration. Half-lives of T and E1S were 1.1 and 0.7 h, respectively, and concentration of T and E1S was similar to that of geldings at 24 h post-castration, which, therefore, should be considered an optimal time to ensure complete castration has occurred.

Transcriptomic analysis reveals the key regulators and molecular mechanisms underlying myometrial activation during equine placentitis†.

The key event in placentitis-induced preterm labor is myometrial activation with the subsequent initiation of labor. However, the molecular mechanisms underlying myometrial activation are not fully understood in the mares. Therefore, the equine myometrial transcriptome was characterized during placentitis (290.0 ± 1.52 days of GA, n = 5) and the prepartum period (330 days of GA, n = 3) in comparison to normal pregnant mares (289.8 ± 2.18 days of GA, n = 4). Transcriptome analysis identified 596 and 290 DEGs in the myometrium during placentitis and the prepartum period, respectively, with 138 DEGs in common. The placentitis DEGs included eight genes (MMP1, MMP8, S100A9, S100A8, PI3, APOBEC3Z1B, RETN, and CXCL2) that are exclusively expressed in the inflamed myometrium. Pathway analysis elucidated that inflammatory signaling, Toll-like receptor signaling, and apoptosis pathways dominate myometrial activation during placentitis. The prepartum myometrium was associated with overexpression of inflammatory signaling, oxidative stress, and 5-hydroxytryptamine degradation. Gene ontology enrichment analysis identified several chemoattractant factors in the myometrium during placentitis and prepartum period, including CCL2, CXCL1, CXCL3, and CXCL6 in common. Upstream regulator analysis revealed 19 potential upstream regulators in placentitis dataset including transcription regulators (E2F1, FOXM1, HIF1A, JUNB, NFKB1A, and STAT1), transmembrane receptors (FAS, ICAM1, SELP, TLR2, and TYROBP), growth factors (HGF and TGFB3), enzymes (PTGS2 and PRKCP), and others (S100A8, S100A9, CD44, and C5AR1). Additionally, three upstream regulators (STAT3, EGR1, and F2R) were identified in the prepartum dataset. These findings revealed the key regulators and pathways underlying myometrial activation during placentitis, which aid in understanding the disease and facilitate the development of efficacious therapies.

Steroid synthesis and metabolism in the equine placenta during placentitis.

Equine placentitis is associated with alterations in maternal peripheral steroid concentrations, which could negatively affect pregnancy outcome. This study aimed to elucidate the molecular mechanisms related to steroidogenesis and steroid-receptor signaling in the equine placenta during acute placentitis. Chorioallantois (CA) and endometrial (EN) samples were collected from mares with experimentally induced placentitis (n = 4) and un-inoculated gestationally age-matched mares (control group; n = 4). The mRNA expression of genes coding for steroidogenic enzymes (3ßHSD, CYP11A1, CYP17A1, CYP19A1, SRD5A1, and AKR1C23) was evaluated using qRT-PCR. The concentration of these enzyme-dependent steroids (P5, P4, 5αDHP, 3αDHP, 20αDHP, 3ß-20αDHP, 17OH-P, DHEA, A4, and estrone) was assessed using liquid chromatography-tandem mass spectrometry in both maternal circulation and placental tissue. Both SRD5A1 and AKR1C23, which encode for the key progesterone metabolizing enzymes, were downregulated (P < 0.05) in CA from the placentitis group compared to controls, and this downregulation was associated with a decline in tissue concentrations of 5αDHP (P < 0.05), 3αDHP (P < 0.05), and 3ß-20αDHP (P = 0.052). In the EN, AKR1C23 was also downregulated in the placentitis group compared to controls, and this downregulation was associated with a decline in EN concentrations of 3αDHP (P < 0.01) and 20αDHP (P < 0.05). Moreover, CA expression of CYP19A1 tended to be lower in the placentitis group, and this reduction was associated with lower (P = 0.057) concentrations of estrone in CA. Moreover, ESR1 (steroid receptors) gene expression was downregulated (P = 0.057) in CA from placentitis mares. In conclusion, acute equine placentitis is associated with a local withdrawal of progestins in the placenta and tended to be accompanied with estrogen withdrawals in CA.

Uterine cervix as a fundamental part of the pathogenesis of pregnancy loss associated with ascending placentitis in mares.

Anatomical and molecular changes in the cervical barrier in women are a fundamental part of the pathogenesis of pregnancy loss associated with chorioamnionitis. However, there is little information regarding changes in the cervix associated with ascending infection in pregnant mares. To better characterize morphological and molecular changes in the cervix during placentitis, we examined full thickness histology and mRNA expression for a number of inflammatory and endocrine factors in the mucosa and stroma of the cervix of mares (n = 5) after experimental induction of placentitis via transcervical inoculation with Streptococcus equi ssp zooepidemicus at approximately 290d of gestation. Gestationally age-matched mares (n = 4) served as controls. Target transcripts included steroid receptors (PGR, ESR1 and 2), OXTR, prostaglandins synthases and receptors (PTGS1, PTGS2, PGES, PGFS, PTGER2 and PTGER4), cytokines (IL1b, IL6, CLCX8, IL10 and TNFα) and acute phase proteins (SAA). Histologically, a marked modification in the cervical epithelia and stroma was characterizing cervicitis. Additionally, the mRNA expression of IL1ß, IL6, CXCL8, SAA and PTGS2 was greater (P < 0.05) in both mucosa and stroma of the inoculated mares; whereas TNFα, IL10 and PGES were upregulated (P < 0.05) only in the cervical mucosa. Progesterone receptor, ESR1 and PTGER4 were upregulated in the cervical stroma of placentitis mares. In conclusion, the cervical response to placentitis was characterized by an upregulation of inflammatory cytokines that was accompanied by induction of PTGS2 and PGES. Further, receptors known to be associated with relaxation of the cervix in other species (ESR1 and PTGER4) were upregulated in the cervical stroma of placentitis mares. These findings indicate that the cervix is not only a physical barrier but that it has an active role in the pathogenesis of ascending placentitis.

Expression Profile of the Chromosome 14 MicroRNA Cluster (C14MC) Ortholog in Equine Maternal Circulation throughout Pregnancy and Its Potential Implications.

Equine chromosome 24 microRNA cluster (C24MC), the ortholog of human C14MC, is a pregnancy-related miRNA cluster. This cluster is believed to be implicated in embryonic, fetal, and placental development. The current study aimed to characterize the expression profile of this cluster in maternal circulation throughout equine gestation. The expression profile of miRNAs belonging to this cluster was analyzed in the serum of non-pregnant (diestrus), pregnant (25 d, 45 d, 4 mo, 6 mo, 10 mo), and postpartum mares. Among the miRNAs examined, 11 miRNAs were differentially expressed across the analyzed time-points. Four of these miRNAs (eca-miR-1247-3p, eca-miR-134-5p, eca-miR-382-5p, and eca-miR-433-3p) were found to be enriched in the serum of pregnant mares at Day 25 relative to non-pregnant mares. To further assess the accuracy of these miRNAs in differentiating pregnant (25 d) from non-pregnant mares, receiver operating characteristic (ROC) analysis was performed for each of these miRNAs, revealing that eca-miR-1247-3p and eca-miR-134-5p had the highest accuracy (AUCROC = 0.92 and 0.91, respectively; p < 0.05). Moreover, eca-miR-1247-3p, eca-miR-134-5p, eca-miR-409-3p, and eca-miR-379-5p were enriched in the serum of Day 45 pregnant mares. Among those miRNAs, eca-miR-1247-3p and eca-miR-409-3p retained the highest accuracy as shown by ROC analysis. GO analysis revealed that these miRNAs are mainly implicated in nervous system development as well as organ development. Using in situ hybridization, we localized eca-miR-409-3p in the developing embryo (25 d) and extra-embryonic membranes (25 and 45 d). In conclusion, the present study is the first to elucidate the circulating maternal profile of C24MC-associated miRNAs throughout pregnancy and to suggest that serum eca-miR-1247-3p, eca-miR-134-5p, and eca-miR-409-3p could be used as pregnancy-specific markers during early gestation (25 and 45 d). Overall, the high abundance of these embryo-derived miRNAs in the maternal circulation suggests an embryo-maternal communication during the equine early pregnancy.

Concentrations of sulphated estrone, estradiol and dehydroepiandrosterone measured by mass spectrometry in pregnant mares.

BACKGROUND: Few studies have provided a longitudinal analysis of systemic concentrations of conjugated oestrogens (and androgens) throughout pregnancy in mares, and those only using immunoassay. The use of liquid chromatography tandem mass spectrometry (LC-MS/MS) will provide more accurate concentrations of circulating conjugated steroids. OBJECTIVES: To characterise circulating concentrations of individual conjugated steroids throughout equine gestation by using LC-MS/MS. STUDY DESIGN: Longitudinal study and comparison of pregnant mares treated with vehicle or letrozole in late gestation. METHODS: Sulphated oestrogens and androgens were measured in mares throughout gestation and mares in late gestation (8-11 months) treated with vehicle or letrozole to inhibit oestrogen synthesis in late gestation. An analytical method was developed using LC-MS/MS to evaluate sulphated estrone, estradiol, testosterone and dehydroepiandrosterone (DHEAS) during equine gestation. RESULTS: Estrone sulphate concentrations peaked by week 26 at almost 60 µg/mL, 50-fold higher than have been reported in studies using immunoassays. An increase in DHEAS was detected from 7 to 9 weeks of gestation, but concentrations remained consistently low (if detected) for the remainder of gestation and testosterone sulphate was undetectable at any stage. Estradiol sulphate concentrations were highly correlated with estrone sulphate but were a fraction of their level. Concentrations of both oestrogen sulphates decreased from their peak to parturition. Letrozole inhibited estrone and estradiol sulphate concentrations at 9.25 and 10.5 months of gestation but, no increase in DHEAS was observed. MAIN LIMITATIONS: Limited number of mares sampled and available for analysis, lack of analysis of 5α-reduced and B-ring unsaturated steroids due to lack of available standards. CONCLUSIONS: Dependent on methods of extraction and chromatography, and the specificity of primary antisera, immunoassays may underestimate oestrogen conjugate concentrations in blood from pregnant mares and may detect androgen conjugates (neither testosterone sulphate nor DHEAS were detected here by LC-MS/MS) that probably peak coincident with oestrogen conjugates between 6 and 7 months of equine gestation.

Inhibin-A and Inhibin-B in stallions: Seasonal changes and changes after down-regulation of the hypothalamic-pituitary-gonadal axis.

The biological function of inhibin is mediated by two heterodimers, inhibin-A and inhibin-B. The relative importance of inhibin-A and -B in male reproductive function varies considerably across species with inhibin-B predominating in many species, whereas inhibin-A appears relatively more important in rams. Research reported to date in stallions has examined total or immunoreactive (ir) inhibin which does not distinguish the two heterodimers. Therefore, the objective of this study was to characterize changes in inhibin-A and inhibin-B concentrations in stallions: 1) across season for a period of one year, and 2) after downregulation of the hypothalamic-pituitary-gonadal (HPG) axis. In Study one, serum samples were obtained monthly from five stallions for a period of one year. Serum concentrations of inhibin-A, inhibin-B, testosterone and estrone sulfate were determined by ELISA. In Study two, stallions were treated with the GnRH antagonist, acyline (n = 4; 330 mg/kg acyline IM) or vehicle control (n = 4; vehicle alone) every five days for 50 days. Plasma concentrations of inhibin-A and -B were determined by ELISA at Days 0, 6, 12, 22, 37, 59, 80, 87 and 104 after initiation of acyline treatment. Testis volume was determined by ultrasonography at weekly intervals. In Study 1, both inhibin-A and inhibin-B showed seasonal changes in concentration with highest concentrations in increasing day length and lowest concentrations in short day lengths. Inhibin-B (overall mean 107.8 ±â€¯4.1 pg/mL) was present at 4.7-fold higher concentrations in serum than inhibin-A (overall mean 23.0 ±â€¯0.7 pg/mL). In Study 2, plasma concentrations of inhibin-B but not inhibin-A were significantly downregulated by administration of the GnRH antagonist, acyline. When the HPG axis was downregulated by acyline, testis volume was strongly correlated with inhibin-B (r = 0.73; P < 0.05) but not inhibin-A (r = 0.22; P = 0.20). In summary, inhibin-B appears to be the predominant form of inhibin in the stallion which undergoes seasonal regulation along with other reproductive parameters and is co-regulated with other endocrine parameters of the HPG axis.

Changes in maternal androgens and oestrogens in mares with experimentally-induced ascending placentitis.

REASONS FOR PERFORMING STUDY: While advanced stages of ascending placentitis can be diagnosed by transrectal ultrasonography and clinical signs, early stages can be missed. Thus, additional tools could enhance assessment of placental health. OBJECTIVES: To characterise peripheral dehydroepiandrosterone sulphate (DHEA-S) and testosterone concentrations in mares carrying normal pregnancies (Study 1) and compare plasma concentrations of DHEA-S, testosterone, oestradiol 17-ß (oestradiol) and oestrone sulphate (OES) in mares with or without placentitis (Study 2). STUDY DESIGN: Longitudinal cohort study of healthy mares (Study 1) and controlled experiment (Study 2). METHODS: In Study 1, mares had serum samples collected from 100 days of gestation to term. In Study 2, pregnant mares (260-280 days gestation) were assigned to a control group or a group with placentitis. Placentitis was induced via intracervical inoculation of Streptococcus equi ssp. zooepidemicus. Blood was collected at inoculation/commencement for control mares (day = 0) and daily for 12 days post inoculation (DPI) or until abortion. Steroid concentrations were determined by immunoassays. Concentrations of steroids in Study 2 were also evaluated relative to days from abortion (DFA -8 days to 0). RESULTS: In Study 1, DHEA-S peaked by 180 days gestation, while testosterone concentrations were progressively increased from Days 100 to 180 with a plateau until ~240 days and a progressive decline until 290 days of gestation. In Study 2, concentrations of DHEA-S and testosterone were not significantly different between groups. There were significant effects of time (oestradiol P = 0.0008, OES P = 0.01) and time-by-group interactions (oestradiol P<0.001, OES P<0.0001) for oestrogen concentrations. For mares with experimental placentitis, concentrations of oestradiol were significantly reduced at -6, -2, -1 and 0 DFA, while OES concentrations were significantly reduced on the day before abortion (0 DFA). CONCLUSIONS: Testosterone and DHEA-S were increased and varied through pregnancy. Oestrogens but not androgens decreased significantly in mares with experimentally-induced ascending placentitis.